Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in the circulation, and in human and animal tissues. Oxysterols that have been identified in human plasma to date include 7α-hydroxycholesterol, 24S-hydroxycholesterol, and 4α- and 4β-hydroxycholesterol, which are present at concentrations ranging from 5-500 ng/ml. These oxysterols have a variety of half-lives in circulation ranging from 0.5-60 hours, and their levels can be altered by aging, drug interventions, and disease processes. Oxysterols may be formed either by autooxidation, as a secondary byproduct of lipid peroxidation, or by the action of specific monooxygenases, most of which are members of the cytochrome P450 family of enzymes. Examples of these enzymes are cholesterol 7α-hydroxylase (CYP7A1) that forms 7α-hydroxycholesterol, cholesterol 25-hydroxylase that forms 25-hydroxycholesterol, cholesterol 24S-hydroxylase (CYP46) that forms 24S-hydroxycholesterol, and others. In addition, oxysterols may be derived from the diet. Cytochrome P450 enzymes are also involved in the further oxidation of oxysterols and their metabolism into active or inactive metabolites that leads to their eventual removal from the system. Since certain oxysterols have potent effects on cholesterol metabolism, their involvement in that process has been widely studied in recent years. In addition, the presence of oxysterols in atherosclerotic lesions has prompted studies of their potential role in the pathogenesis of this disorder. A role for specific oxysterols has been implicated in various physiologic processes including cellular differentiation, inflammation, apoptosis, and steroid production. Moreover, due to the abundance of cholesterol in living organisms, the prooxidant nature of our environment, and the multitude of enzymatic and non-enzymatic pathways for their production, it would not be surprising to find that oxysterols play additional, as yet unidentified, roles in biological systems.
Recently, several reports have noted the possible role of oxysterols in cellular differentiation. Specific oxysterols induce the differentiation of human keratinocytes in vitro, while monocyte differentiation can be induced by the oxysterol 7-ketocholesterol. Our previous reports have shown that specific oxysterols induce the differentiation of pluripotent mesenchymal cells into osteoblastic cells, while inhibiting their differentiation into adipocytes. Differentiation of keratinocytes by oxysterols is mediated by the nuclear hormone receptor, liver X receptor β (LXRβ). LXRα and LXRβ, initially identified as orphan nuclear receptors, act as receptors for oxysterols. However many of the effects of oxysterols are mediated by LXR-independent mechanisms. These include their effects on mesenchymal cells, since activation of LXR by specific LXR ligands inhibited, rather than stimulated, the osteogenic differentiation of mesenchymal cells. Furthermore, MSC derived from LXR null mice were able to respond to osteogenic oxysterols as well as their wild type counterparts. Additional oxysterol binding proteins have been reported that can regulate the activity of signaling molecules such as mitogen-activated protein kinase (MAPK).
Hedgehog molecules have been shown to play key roles in a variety of processes including tissue patterning, mitogenesis, morphogenesis, cellular differentiation and embryonic developments. In addition to its role in embryonic development, hedgehog signaling plays a crucial role in postnatal development and maintenance of tissue/organ integrity and function. Studies using genetically engineered mice have demonstrated that hedgehog signaling is important during skeletogenesis as well as in the development of osteoblasts in vitro and in vivo. In addition to playing a pro-osteogenic role, hedgehog signaling also inhibits adipogenesis when applied to pluripotent mesenchymal cells, C3H-10T ½.
Hedgehog signaling involves a very complex network of signaling molecules that includes plasma membrane proteins, kinases, phosphatases, and factors that facilitate the shuffling and distribution of hedgehog molecules. Production of hedgehog molecules from a subset of producing/signaling cells involves its synthesis, autoprocessing and lipid modification. Lipid modification of hedgehog, which appears to be essential for its functionality, involves the addition of a cholesterol molecule to the C-terminal domain of the auto-cleaved hedgehog molecule and palmitoylation at its N-terminal domain. Additional accessory factors help shuttle hedgehog molecules to the plasma membrane of the signaling cells, release them into the extracellular environment, and transport them to the responding cells.
In the absence of hedgehog molecules, Patched (Ptch), present on the plasma membrane of the responding cells, keeps hedgehog signaling in a silent mode by inhibiting the activity of another plasma membrane associated signal transducer molecule, Smoothened (Smo). In the presence of hedgehog, the inhibition of Smo by Ptch is alleviated and Smo transduces the signal for the regulation of transcription of hedgehog-regulated genes. This transcriptional regulation in part involves the Ci/Gli transcription factors that enter the nucleus from the cytoplasm after a very intricate interaction between the members of a complex of accessory molecules that regulate Gli and its conversion from a 75 kd transcriptional repressor to a 155 kd transcriptional activator. The details of this highly complex signaling network have been extensively reviewed. (Cohen (2003) Am J Med Gen 123A. 5-28; Mullor et al. (2002) Trends Cell Bio 12, 562-569).